System, substrate plate and incubation device for conducting bioassays

ABSTRACT

A system for conducting bioassays comprises a substrate plate with wells, and an incubation device for holding the plate. The substrate plate comprises a microplate with an array of wells arranged in rows and columns, wherein the bottom of each well is a microarray substrate having oriented flow-through channels. The incubation device comprises an incubation chamber fox holding the microplate and a cover for sealing the incubation chamber. The incubation device has a heat block with array of openings, each opening adapted to receive a well of the microplate. A sealing gasket is provided for individually sealing each well of the microplate.

The present invention relates to a system for conducting bioassays,comprising a substrate plate with a number of wells, and an incubationdevice for holding the plate. The invention further relates to asubstrate plate with wells, and to an incubation device for such asystem.

WO 01/19517 of the same applicant discloses a system with an analyticaltest device comprising a substrate such as a metal oxide membrane havingthrough-going oriented channels. Such membranes have oriented channelswith well controlled diameter and advantageous chemical surfaceproperties. When used in a bioassay the channels in at least one area ofthe surface of the metal oxide membrane are provided with a firstbinding substance capable of binding to an analyte. According to apreferred embodiment the metal oxide membrane is comprised of aluminiumoxide. Reagents used in these bioassays are immobilized in the channelsof the substrate and the sample fluid will be forced through thechannels to be contacted with the reagents.

This known analytical test device is composed of a plastic support withan encapsulated substrate layer. Openings in the plastic support definewells with a certain diameter, said wells exposing the substrate, andthe area of the substrate exposed in the well being provided with atleast one binding substance specific for at least one analyte. An amountof sample fluid is added to one or more of the wells of the device, theamount of added sample fluid being calculated on the basis of thedimensions of the wells and the substrate. An alternating flow isgenerated through the substrate in the wells whereby the liquid volumeof sample fluid is forced to pass through the channels in the substratefrom the upper side of the substrate to the lower side of the substrateand back at least one time, under conditions that are favorable to areaction between an analyte present in the sample and the bindingsubstances. Any signal generated in any of the wells is read and fromsaid signals the presence, amount, and/or identity of said one or moreanalytes are determined. When the heat block of the incubator device iscovered by a transparent material, such as a glass cover, the wells canbe analyzed and the reading signal can be determined through the heatblock.

Improvements of this known system are described in international patentapplications PCT/EP02/02446, PCT/EP02/02447 and PCT/EP02/02448 of thesame applicant. The known system is not suitable for high throughputscreening, as it is not automation-friendly and the number of tests inone parallel processing cycle is restricted.

The invention aims to provide a system of the abovementioned type withimproved high throughput screening capabilities allowing parallelprocessing of a large number of arrays in automated robotic platforms.

According to the invention a system is provided, wherein the substrateplate comprises a microplate with an array of wells arranged in rows andcolumns, wherein the bottom of each well is a microarray substratehaving oriented flow-through channels, and in that the incubation devicecomprises an incubation chamber for holding the microplate and a coverfor sealing the incubation chamber, said incubation device having a heatblock with array of openings, each opening adapted to receive a well ofthe microplate, wherein a sealing gasket is provided for individuallysealing each well of the microplate.

In this manner a system is obtained with a microplate with wells whichcan be made according to a SBS standard format allowing the use ofstandard screening instrumentation, especially in automated roboticplatforms. Using for example a microplate with an array of ninety-sixwells allows a parallel processing of a large number of microarraysresulting in a very efficient high throughput screening.

The invention further provides a microplate, comprising an array ofwells arranged in rows and columns, wherein the bottom of each well is amicroarray substrate having oriented flow-through channels.

The invention also provides an incubation device to be used in thesystem of the invention.

Finally, the invention provides anpparatus for conducting highthroughput screening tests, comprising a system of the invention, adevice for linearly moving the incubation device along a plurality ofstations including a station for loading a microplate into theincubation device, a station for dispensing a liquid into the wells ofthe microplate, and a reading station for individually illuminating eachsubstrate of the microplate, wherein a device is provided for moving theincubation device with the microplate with respect to the readingstation in mutually perpendicular directions.

The invention will be further explained by reference to the drawings inwhich embodiments of the system, the microplate and the incubationdevice of the invention are schematically shown.

FIG. 1 shows a top view of an embodiment of the system of the invention.

FIG. 2 is a side view of the system of FIG. 1, wherein the incubationdevice, the cover and the microplate are separately shown.

FIG. 3 shows a side view of the system of FIG. 1, wherein the wells ofthe microplate are located within the openings of the heat block of theincubation chamber.

FIG. 4 is a side view of the system of FIG. 1, wherein the cover is inits closed position.

FIG. 5 shows an apparatus for performing bioassays using the system ofthe invention.

Referring to the drawings, there is shown a system for performingbioassays, preferably high throughput screening tests. The systemcomprises a microplate 1 as substrate plate, the microplate 1 having anarray of wells 2 arranged in rows and columns, as can be seen in FIG. 1.In the embodiment shown, the microplate 1 comprises ninety-six wellsarranged in eight rows and twelve columns. Of course other arrayarrangements are possible, for example with 8, 12, 24, 48, 384 or 1536wells. As schematically shown in the side views of the system of FIGS.2-4, the bottom of each well 2 is provided by a microarray substrate 3.The substrates 3 are located substantially in the sam virtual plane.

Each substrate 3 is made of a porous flow-through metal oxide membrane.The substrate 3 is preferably an aluminium oxide having a large numberof through-going channels oriented mainly perpendicular to the upper andlower services of the substrate. Preferably the channels are capillarychannels. In a practical embodiment of the substrate 3, the internaldiameter d of the substrate can be 5 mm, wherein the channels may have aspacing of approximately 150-200 nm. A binding substance can be bound tothe substrate in groups of channels at a spacing of 200 μm. Such a groupof channels can be indicated as a spot or spot area. Each substrate 3may have 300-400 spots or more. For a further description of thesubstrate material reference is made to the above-mentionedinternational patent application WO 01/19517. It will be understood thatthe number of wells, the number of spots and the dimensions arementioned by way of example only and may be varied as desired.

In a preferred embodiment the wells 2 have a conical shape as shown inthe drawings. However, the wells 2 may have a different shape. Theconical shape of the wells 2 optimizes the imaging characteristics ofthe microplate 1, i.e. reduction of scattering and reflection of lightand enablement of darkfield imaging. The microplate 1 has a skirt 4,wherein the lower side of the skirt 4 is located in the same virtualplane as the substrates 3 or is located at a higher level. Suchdimensions of the skirt 4 allows an on-the-fly spotting of thesubstrates 3 of the microplate 1. The microplate 1 is made of a suitableplastic material, e.g. LCP, TOPAS or polypropylene, but it can also bemade out of other suitable materials such as glass or silicon. Thematerial used must be chemically resistant and heat resistant upto 120°C., robot compatible, optically compatible, i.e. flat and minimalautofluorescence. Further the material should have minimal bindingproperties for labeled biomolecules. Preferably the microplate materialis black to minimize autofluorescence and refractive back scattering oflight. As an alternative it is possible to provide the microplate 1 witha coating to obtain the desired non-reflective properties.

The substrates 3 are incorporated into the wells 2 by moulding, glueing,thermal bonding or any other suitable method. The substrates 3 are flatand are preferably located in the same virtual plane, i.e. are parallelto a virtual plane within a distance less than 100 μm.

The system further comprises an incubation device 5 providing anincubation chamber 6 for holding the microplate 1 and a cover 7 forsealing the incubation chamber 6. The incubation device 5 has a heatblock 8 with an array of openings 9, each opening having a conical shapecorresponding to the shape of the wells 2. The conical shape of thewells 2 provides a self-centering effect during positioning of themicroplate 1 in the incubation device 5. The maximum thickness of theheat block 8 corresponds with the depth of the wells 2 of the microplate1. In this manner the substrates 3 of the wells 2 are either projectingout of the heat block 8 or aligned flush with the lower surface of theheat block 8. Thereby a sample fluid attached to the lower surface of asubstrate 3 cannot contaminate the heat block 8.

Each well is received within an opening 9, so that the outer wall of awell 2 of the microplate 1 is fitted within the inner wall of thecorresponding opening 9. In this manner an optimum heat transfer fromthe heat block 8 to the wells 2 is obtained.

The incubation device 5 has a circumferential wall 10 and a bottom wall11, wherein the heat block 8, the circumferential wall 10 and the bottomwall 11 enclose an air chamber 12 having a connection 13 for an externalvacuum/pressure system not shown. Further, the air chamber 12 has adrain connection 14. The drain connection 14 can be closed by means of avalve not shown.

The incubation device 5 is preferably made of a metal and is providingwith a heating element to control the temperature of the incubationchamber and thereby of sample fluids provided in the wells 2 of amicroplate 1 received in the incubation chamber. The heating element canbe made as a heating block containing one or more Peltier elements. Asan alternative heat may be transferred to the incubation chamber via awater bath.

As shown in FIGS. 2-4, a sealing gasket 15 is provided on the lower sideof the circumferential wall of the cover 7. As an alternative the gasketcould be provided on the upper side of the circumferential wall 10 ofthe incubation device 5. This sealing gasket 15 seals the incubationdevice 5 when the cover 7 is in the closed position of FIG. 4. The airchamber 12 is then closed in an air-tight manner. A further sealinggasket 16 is provided, having circular openings 17 with a diametercorresponding to the diameter of the openings 9 at the surface of theheat block 8. Preferably the sealing gasket is sealingly fixed on theinner side of the cover 7. When the cover is in its closed position thegasket 16 sealingly engages the upper side of the microplate 1. In viewof the shape of the sealing gasket 16 each well 2 of the microplate 1 isindividually sealed with respect to the other wells 2 and theenvironment.

The cover 7 is preferably transparent and is made of glass, for example.The cover 7 can be provided with a heating element, for example byincorporating transparent electrical wires in the cover material. As analternative a heating element having the same shape as the heat block 8could be used for heating the cover. The cover 7 can be heated in thismanner to prevent condensation during conducting a high throughputscreening test. The transparency of the cover allows a real timemeasurement to be made from above using a CCD system or a suitableoptical scanner.

During operation, the pressure in the incubation device can becontrolled by a vacuum/pressure system connected to the connection 13.To perform high throughput screening bioassays, one or more samplefluids are provided in the wells 2 and the microplate 1 is inserted intothe incubation chamber 6. The cover 7 is brought in its closed positionas shown in FIG. 4 and the pressure within the air chamber 12 iscontrolled. A low pressure in the chamber 12 creates a pressuredifference over the substrate 3, whereby the sample fluid is forcedthrough the channels of the substrate 3, thereby creating a low pressurewithin the wells 2. By removing the low pressure in the chamber 12, thesample fluid is automatically forced back through the channels of thesubstrates 3 into the wells 2. Of course, it is possible to create ahigh pressure in the chamber 12 to force the sample fluid through thechannels into the wells 2 more rapidly. By alternatingly creating a lowpressure in the chamber 12 and removing the low pressure, the samplefluids are forced through the channels of the substrate a number oftimes. The individual sealing of each of the wells 2 shows the advantagethat a malfunction of one of the substrates 3, which prevents thecreation of a pressure difference over the substrate, will not preventnormal use of the other substrates 3.

The imaging of the bioassay is done from above through the transparentcover 7 using a CCD camera for example. This allows a real time kineticmeasurement. The height h of the chamber 12 is such that a standardmicroplate with a corresponding array of wells can be located in thechamber 12 to collect filtrate from the microplate 1. The chamber 12 canfurther be used as a humidifying chamber by releasing a small amount ofliquid in the chamber. Thereby evaporation of sample liquid issignificantly reduced at elevated temperatures and during extendedoperations. Flow-through washing of the substrates 3 is possible. Thedrain connection 14 allows the disposal of the washing liquids.

Preferably the incubation device 5 is part of an apparatus forconducting high throughput screening tests, an embodiment of which isshown in a very schematical manner in FIG. 5. According to FIG. 5, theapparatus comprises a platform 18 supporting a device 19 for linearlymoving the incubation device S. By means of the device 19, theincubation device 5 can be positioned with great accuracy in theX-direction at the locations A-D indicated in FIG. 5. In location A, theincubation device 5 is in a position for loading a microplate 1 into thedevice 5 by means of a robot. A dispenser station 20 is located inposition B. This station 20 is adapted to dispense a washing liquid intothe wells 2 of the microplate 1. After treatment of the microplate 1 atthe location B, the incubation device 5 is moved into position C, wherea further treatment of the microplate 1 is possible. For this treatmenta special cover 21 is placed on the incubation device 5. This cover 21is provided with an array of needles 22 corresponding with the array ofwells 2 of the microplate 1. Through these needles 22, the pressurewithin the wells 22 above the substrates 3 can be increased tofacilitate the flow of the sample liquid through the substrates 3.Further, air can be blown on the substrates 3 through these needles 22.

A reading station 24 is provided at the location D. In order to readeach of the substrates 3 the platform 18 is moveable in X andY-direction. In this manner each substrate 3 can be illuminated by aradiation source of the reading station 24 and the fluorescence is readby means of a CCD camera of the reading station 24. Instead of theillumination shown in FIG. 5, a so-called dark field illumination, i.e.illumination under an angle with respect to the substrate, is alsopossible.

Preferably, a microplate 1 is used meeting the standard format asproposed by the Society for Biomolecular Screening (SBS) formicroplates. This allows the use of current industry standards forscreening applications and screening instrumentation, especially the useof automated robotic platforms In this manner, the system as describedcan be used in applications such as genotyping, including SNP analysis,gene expression profiling, proteomics, ELISA-based bioassays,receptor-ligand binding bioassays and enzyme kinetic bioassays.

It will be understood that the system of the invention allows parallelprocessing of a large number of microarrays. A sequential fluorescentdetection of the microarrays by imaging per well is facilitated by theflatness and location of the substrates in the same virtual plane.Further the dimensions of the wells, in particular the conical shape ofthe wells allows the sequential fluorescent detection. The system isadapted to automation and is robot compatible. The individual sealing ofthe wells shows the advantage that in case of substrate breakage thereis no interference of the control of the pressure variation at the othersubstrates. The microplate 1 allows for an on the fly spotting of thebinding agents.

The invention is not restricted to the above-described embodiment whichcan be varied in a number of ways within the scope of the claims.

1. System for conducting bioassays, comprising a substrate plate with anumber of wells, and an incubation device for holding the plate,characterized in that the substrate plate comprises a microplate with anarray of wells arranged in rows and columns, wherein the bottom of eachwell is a microarray substrate having oriented flow-through channels,and in that the incubation device comprises an incubation chamber forholding the microplate and a cover for sealing the incubation chamber,said incubation device having a heat block with array of openings, eachopening adapted to receive a well of the microplate, wherein a sealinggasket is provided for individually sealing each well of the microplate.2. System according to claim 1, wherein the incubation device comprisesa circumferential wall, wherein a sealing gasket is provided on theupper side of said circumferential wall, said sealing gasket beingadapted to sealingly engage the lower side of the microplate.
 3. Systemaccording to claim 1, wherein the maximum thickness of the incubationdevice heat block corresponds with the depth of the wells of themicroplate, wherein preferably the circumferential wall of each openingis adapted to contact the outer wall of a well of the microplate. 4.System according to claim 3, wherein the wells of the microplate and theopenings of the heat block are conically shaped.
 5. System according toclaim 1, wherein the heat block, the circumferential wall and a bottomwall of the incubation device enclose an air chamber having a connectionfor an external vacuum/pressure system and a drain connection.
 6. Systemaccording to claim 1, wherein the cover is transparent.
 7. Systemaccording to claim 1, wherein the cover is provided with a heatingelement.
 8. System according to claim 1, wherein the incubation deviceis provided with a heating element.
 9. System according to claim 1,wherein the substrate is made of a metal oxide, preferably an aluminiumoxide.
 10. Microplate, comprising an array of wells arranged in rows andcolumns, wherein the bottom of each well is a microarray substratehaving oriented flow-through channels.
 11. Microplate according to claim10, wherein each well has a conical shape.
 12. Microplate according toclaim 10, wherein at least the upper surface of the microplate and theinner side of the wells is non-reflecting.
 13. Microplate according toclaim 10, comprising a skirt having a lower side, wherein the substratesof the wells are substantially located in the same virtual plane and thelower side of the skirt is located in the same virtual plane or at ahigher level.
 14. Microplate according to claim 10, wherein allsubstrates are substantially located in the same virtual plane. 15.Microplate according to claim 10, wherein the substrates areincorporated in the plate by moulding, glueing, thermal bonding or thelike.
 16. Microplate according to claim 10, wherein the substrate ismade of a metal oxide, preferably an aluminium oxide.
 17. Incubationdevice for a system according to claim
 1. 18. Apparatus for conductinghigh throughput screening tests, comprising a system according to claim1, a device for linearly moving the incubation device along a pluralityof stations including a station for loading a microplate into theincubation device, a station for dispensing a liquid into the wells ofthe microplate, and a reading station for individually illuminating eachsubstrate of the microplate, wherein a device is provided for moving theincubation device with the microplate with respect to the readingstation in mutually perpendicular directions.